1. Field of the Invention
This invention relates generally to a method of separating and purifying Factor IX and other vitamin K-dependent proteins including Factor II, Factor VII, Factor X, prothrombin, Protein C, and Protein S. More specifically, high purity protein is separated by a chromatographic adsorption and concentration technique from plasma or concentrate.
2. Background of the Invention
Blood coagulation factors play a vital role in the normal coagulation mechanism. For instance, patients with a deficiency of Factor IX exhibit severe bleeding problems ("Hemophilia B"). It would be desirable to be able to isolate substantial quantities of Factor IX and other vitamin K-dependent proteins for therapeutic administration, as well as for scientific study, and several processes directed to this objective have been described in the literature. The process of the present invention surpasses these processes in providing a combination of high yield of the desired protein with high purity, and particularly with relatively low contamination by other clotting factors. In addition, the prior art processes require a large number of steps each of which is time-consuming and introduces losses of protein into the overall process.
One such process is that of S. P. Bajaj, et al., "A Simplified Procedure for Purification of Human Prothrombin, Factor IX and Factor X", in Preparative Biochemistry, 11 (4), 397-412 (1981). This process subjects pooled plasma to adsorption onto and elution from barium citrate, ammonium sulfate fractionation, DEAE-Sephadex chromatography, and heparin-agarose chromatography in a sodium citrate buffer at pH 7.5, to separate prothrombin, Factor X, and Factor IX. The reported yield of Factor IX was about 35%, at a specific activity of 80-220 units/mg. Another process is that of J. P. Miletich, et al., "Purification of Human Coagulation Factors II, IX, and X Using Sulfated Dextran Beads" in Methods in Enzymology, Vol. 80, pp. 221-228. Fresh frozen plasma is thawed, and subjected to a sequence of precipitation with barium citrate, resuspension in Tris-HCl with diisopropylfluorophosphate, precipitation with ammonium sulfate, chromatography on DE-52 cellulose, and application of selected pooled fractions to sulfated dextran beads. Following concentration, the fraction containing the Factor IX is said to be contaminated with as much as 1 wt. % of Factor X. B. Osterud, et al. in "Human Blood Coagulation Factor IX". J. Biological Chemistry, Vol. 253, No. 17, pp. 5946-5951 (1978), describe the purification of Factor IX by a sequence of adsorption onto and elution from barium sulfate, DEAE-cellulose batch chromatography, polyacrylamide gel electrophoresis, and heparin-agarose affinity chromatography. The Factor IX activity was said to be 207 units/mg, though the yield of Factor IX was not stated.